Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration

Abstract

Penicillin G acylase from Bacillus megaterium (BmPGA) is currently used in the pharmaceutical industry as an alternative to PGA from Escherichia coli (EcPGA) for the hydrolysis of penicillin G to produce 6‐aminopenicillanic acid (6‐APA), a penam nucleus for semisynthetic penicillins. Despite the significant differences in amino‐acid sequence between PGAs from Gram‐positive and Gram‐negative bacteria, a representative PGA structure of Gram‐positive origin has never been reported. In this study, crystallization and diffraction studies of BmPGA are described. Poor diffraction patterns with blurred spots at higher resolution were typical for BmPGA crystals cryocooled after a brief immersion in cryoprotectant solution. Overnight soaking in the same cryo‐solution substantially improved both the mosaicity and resolution limit through the establishment of a new crystal‐packing equilibrium. A crystal of BmPGA diffracted X‐rays to 2.20 Å resolution and belonged to the monoclinic space group P21 with one molecule of BmPGA in the asymmetric unit.

An evaluation of adhesive sample holders for advanced crystallographic experiments

“…Dehydration is one way of exploring the further diffraction potential of a crystal and can lead to dramatic improvement of the crystal diffraction quality (Russo Krauss et al, 2012). In some cases, reducing the solvent content by dehydration has enabled atomic resolution structures to be obtained from previously poorly diffracting crystals (Heras et al, 2003;Yap et al, 2007;Sam et al, 2006;Haebel et al, 2001;Koch et al, 2004;Rojviriya et al, 2011;Pauwels et al, 2005;Stocker et al, 2005). Changing the crystal solvent content may alter the unit-cell parameters and in some cases even lead to major lattice rearrangements including space-group changes (Jenni & Ban, 2009;Bailly et al, 2009).…”